Extracellular Vesicle Capture and microRNA Detection

Abstract

Cancer is one of the leading causes of death in the United states, and there has been much focus on earlier detection through the discovery of novel, easily accessible biomarkers via liquid biopsies. Extracellular vesicles have shown promise as a noninvasive biomarker for disease diagnosis and monitoring, and have become a treasure trove of information because they have been found to carry proteins, DNA, mRNA and microRNA as well surface markers indicative of the their cell origin. Thus developing methods to profile extracellular vesicles and interrogate the contents of these vesicles is a growing area of research and has the potential to develop into a non-invasive diagnostic platform, a liquid biopsy.

The aim of this thesis is to develop a system to capture extracellular vesicles and profile the miRNA patterns present within them. First, we develop various amplification strategies in hydrogel particles for microRNA detection, including a colorimetric detection platform that can be translated to point-of-care settings for a liquid biopsy. We also explored other amplification strategies for increased miRNA detection sensitivity including precipitation-based enzymatic signal amplification and strand displacement amplification.

Then we develop methods for extracellular vesicle lysis and miRNA detection using a one-pot lysis and miRNA capture method. Extracellular vesicles were isolated from matched diseased and normal donor serum. Using rolling circle amplification, we performed multiplexed miRNA detection and quantification from serum extracellular vesicles. Calibration curves using rolling circle amplification were used to determine miRNA copy number estimates in agreement with other studies in literature with absolute quantification.

Finally, we tune hydrogel particle porosity and use novel functionalization techniques to capture extracellular vesicles based on their surface markers. We explored the use of the thiol-acrylate Michael addition reaction for antibody conjugation and optimized it for extracellular vesicle capture. Using these porous, antibody functionalized hydrogel particles, we captured breast cancer serum and match healthy serum extracellular vesicles using two surface markers, paving the way for multiplexed extracellular vesicle surface marker characterization. Porous hydrogel particles have the potential to considerably enhance the workflow for exosome capture and profiling experiments, through multiplexing, fewer sample preparation requirements, and customizable nature, hence furthering extracellular vesicle research.

Incorporating the insights from the MIT Sloan Management program, the commercialization potential and current market landscape for extracellular vesicles was analysed. Extracellular vesicles have shown tremendous potential in the field of therapeutics, diagnostics, and for furthering academic research. The market study shows that the field is growing rapidly, with continued investment from venture capital for new companies, as well as corporate acquisitions by legacy players as they look to enter the field.

The work presented in this thesis employs the various benefits of hydrogels for biomolecule detection, namely their biocompatibility, solution-like kinetics, nonfouling nature, and tunable chemistry. We believe that this work can be leveraged to improve upon and develop new technologies for extracellular vesicle capture and analysis, leading to more insights into this promising biomarker, eventually leading to earlier and more accurate diagnosis of disease.